Divergent Plasmodium kinases drive MTOC, kinetochore and axoneme organisation in male gametogenesis

CDPK4, SRPK1, and MAP2 have different spatiotemporal locations during schizogony and male gametogenesis

Despite several studies highlighting the essential roles of CDPK4, SRPK1, and MAP2 in male gametogenesis in P. berghei (Billker et al, 2004; Tewari et al, 2010), little is known about their location during the endomitotic stages of cell division (i.e., schizogony, sporogony, and male gametogenesis). To examine the spatiotemporal expression of CDPK4, SRPK1, and MAP2, we generated transgenic P. berghei parasite lines expressing the genes modified to code for a C-terminal GFP-tag. An in-frame gfp coding sequence was inserted at the 3′ end of the endogenous gene locus (cdpk4—PBANKA_0615200; srpk1—PBANKA_0401100; map2—PBANKA_0933700), using single-crossover homologous recombination (Fig S1A, Table S1 for primers), and successful insertion was confirmed by diagnostic PCR (Fig S1B, Table S1). Western blot analysis of schizont (for CDPK4 and SRPK1) and gametocyte (for MAP2 as MAP2 seems to be barely expressed in asexual blood stages) protein extracts, using an anti-GFP antibody, revealed major bands at 87.6 kD for CDPK4-GFP, 180.7 kD for SRPK1-GFP, 88.1 kD for MAP2-GFP, and 27 kD for unfused GFP (WT-GFP), respectively; the expected sizes for each. Each GFP-tagged PK was also observed by fluorescence in activated gametocytes (Fig S1C).

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Figure S1. C-terminal GFP tagging of the male-specific protein kinases.

(A) Schematic representation for 3′-tagging of each endogenous P. berghei PK gene with gfp via single homologous recombination. Primers 1+2 used for diagnostic PCR are indicated. (B) For each PK are shown: (left) diagnostic integration PCR (IntPCR) showing band at the expected size, confirming successful integration of the tagging construct; (right) Western blot (WB) analysis using an anti-GFP antibody against control GFP (WT-GFP) and transgenic parasite protein showing bands of 27 kD for WT-GFP and of 87.6, 180.7, and 88.1 kD for CDPK4-GFP, SRPK1-GFP, and MAP2-GFP, respectively (arrows). (C) Images showing expression of the WT-GFP and the GFP-tagged PKs in male gametocytes post-activation. Merge shows Hoechst (DNA, blue) and GFP (green) signals. Scale bar = 5 μm. (D) Additional images of male gametocytes at the late stage, which have more than three CDPK4-GFP foci. Green arrowheads show the CDPK4-GFP fluorescence foci. Scale bar = 5 μm.

Source data are available for this figure.

Source Data for Figure S1[LSA-2024-03056_SdataFS1.pdf]

Live-cell fluorescence imaging of P. berghei asexual blood stages revealed a diffuse cytoplasmic distribution of CDPK4-GFP in both schizonts and merozoites. In contrast, SRPK1-GFP showed weak diffuse cytoplasmic distribution with a strong focus at the location of the nuclear pole and towards the apex of the forming or mature merozoites (Fig 1A). In contrast, no MAP2-GFP fluorescence was observed in asexual development. During male gametogenesis, CDPK4-GFP had a diffuse location in both cytoplasm and nucleus, along with a distinct focus in the cytoplasm six min post-activation (Fig 1B, arrowheads). At the early stage of male gametogenesis (30 s to 6 min post-activation), up to two foci of CDPK4-GFP were observed, whereas at the late stage (>6 min post-activation), more than three foci were sometimes observed (Fig S1D, arrowheads). SRPK1-GFP was located in the cytoplasm and was largely excluded from the nucleus, confirming previous observations in Plasmodium falciparum (Pf; Dixit et al, 2010; Kumar et al, 2021). MAP2-GFP is preferentially located in the nucleus in activated male gametocytes (Fig 1B).

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Figure 1. Fluorescence localisation in PK-tagged lines during asexual and sexual stages.

(A) Localisation of each GFP-tagged PK in schizonts or merozoites. DIC: differential interference contrast. Merge shows Hoechst (DNA, blue) and GFP (green) signals. Scale bar = 5 μm. (B) Localisation of each GFP-tagged PK in activated gametocytes 6 min post-activation. Merge shows Hoechst (DNA, blue) and GFP (green) signals. Green arrowheads indicate CDPK4-GFP concentrated focus in the cytoplasm. Scale bar = 5 μm.

Because these PKs are essential for axoneme development and have specific locations in the parasite cell (in particular, the distinct CDPK4-GFP focus in male gametocytes), we examined their co-localisation with markers of MTOC biology, including chromosome segregation (kinetochore protein, NDC80; Zeeshan et al, 2020) and basal body formation (cytoplasmic axonemal protein, Kinesin-8B; Zeeshan et al, 2019), which were C-terminally tagged with mCherry (mCh). No co-localisation was observed between CDPK4-GFP, SRPK1-GFP, or MAP2-GFP and NDC80-mCh during male gametogenesis (Fig 2A), or between SRPK1-GFP and NDC80-mCh during schizogony (Fig S2A), suggesting that these PKs do not directly interact with kinetochores during chromosome condensation. However, during male gametogenesis, SRPK1-GFP appeared to weakly co-localise with Kinesin-8B-mCh in some areas (Figs 2B and S3). CDPK4-GFP did not co-localise with Kinesin-8B-mCh at any point (Fig 2B). This was surprising, because altered Kinesin-8B phosphopeptide abundance has been observed in cdpk4 mutants (Fang et al, 2017; Kumar et al, 2021). A MAP2-GFP/Kinesin-8B-mCh cross was not generated because these proteins are primarily located in different subcellular compartments.

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Figure 2. Co-localisation of PKs with kinetochore and basal body/axoneme markers.

(A) Co-localisation of each GFP-tagged PK with mCherry (mCh)-tagged NDC80 in gametocytes, 1.5–3 min post-activation. DIC: differential interference contrast. Merge shows Hoechst (DNA, blue), NDC80-mCh (magenta), and GFP (green). Scale bar = 5 μm. (B) Co-localisation of each GFP-tagged PK with mCh-tagged Kinesin-8B in gametocytes 2–8 min post-activation. Merge shows Hoechst (DNA, blue), Kinesin-8B-mCh (magenta), and GFP (green). Scale bar = 5 μm.

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Figure S2. Co-localisation of SRPK1-GFP with kinetochore and centrin markers during asexual blood stages.

(A) Localisation of GFP-tagged SRPK1 crossed with mCh-tagged NDC80 in blood-stage schizonts or merozoites. DIC: differential interference contrast. Merge shows Hoechst (DNA, blue), NDC80-mCh (magenta), and SRPK1-GFP (green). The time on the right indicates the duration within the schizont culture medium. Scale bar = 5 μm. (B) Immunofluorescence microscopy using a GFP-SRPK1 cell line with anti-GFP and anti-centrin or anti-α-tubulin antibodies. Merge shows Hoechst (DNA, blue), anti-centrin or anti-α-tubulin staining (magenta), and anti-GFP staining (green). Arrowheads show the fluorescence foci. Scale bars = 2 μm. The location of SRPK1 and each protein marker is summarised in the schematic diagram on the right.

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Figure S3. Co-localisation of SRPK1-GFP and Kinesin-8B-mCh.

Analysis of GFP-tagged SRPK1 and mCh-tagged Kinesin-8B, co-expressed in gametocytes at 8 min post-activation. Merge shows Hoechst (DNA, blue), Kinesin-8B-mCh (magenta), and GFP (green). Scale bar = 5 μm. Enlarged images of the yellow squares are shown below each corresponding image. The graphs on the right show plots of normalised fluorescence intensity (with the maximum fluorescence intensity in each measurement taken as 1) along the yellow dotted lines in the enlarged images. GFP and mCherry signals are represented by green and magenta lines, respectively.

CDPK4, SRPK1, and MAP2 interact with each other and key regulators of male gametogenesis

Previous studies have highlighted differential protein phosphorylation, detected by altered abundance of phosphopeptides in protease digests, of cellular proteins associated with distinct cell cycle events in Pbcdpk4 and Pbsrpk1 deletion mutants (Fang et al, 2017; Invergo et al, 2017). In addition, SRPK1 was suggested to be phosphorylated in a CDPK4-dependent manner (Invergo et al, 2017). Fang et al (2017) identified CDPK4-interacting proteins and CDPK4-dependent phosphorylation, using immunoprecipitation and mass spectrometry–based phosphoproteomics. However, the protein substrates of SRPK1 and MAP2 during male gametogenesis are largely unknown. We analysed the interactome of each PK in P. berghei gametocyte lysates to identify interacting partners of each PK, which are potential substrates and may regulate male gametogenesis.

In triplicate experiments, we immunoprecipitated with anti-GFP antibody CDPK4-GFP, SRPK1-GFP, MAP2-GFP, and WT-GFP (as a control) from lysates of cells, 6 min post-activation of gametocytes. Immunoprecipitates were digested, and the resultant peptides were analysed by mass spectrometry to identify component proteins. Potential interacting partners for each PK were identified as those with unique peptide sequences present in all three biological replicates, but completely absent from WT-GFP control samples.

A total of 108, 201, and 162 proteins were immunoprecipitated together with CDPK4-GFP, SRPK1-GFP, and MAP2-GFP, respectively (Fig 3A, Tables 1, S2, S3, and S4). Gene Ontology (GO) analysis of CDPK4- and MAP2-GFP partners highlighted several components of the replisome and DNA replication (Fig 3B and D, Tables S5, S6, and S7). Of note, in the CDPK4-GFP line all five known Plasmodium origin recognition complex (ORC) subunits and CDC6 (components of the pre-replicative complex; Chou et al, 2021) were present (Tables 1 and S2), along with the catalytic subunits of DNA polymerase alpha and epsilon. However, no minichromosome maintenance (MCM) proteins were detected, unlike in the MAP2-GFP samples, in which MCM3–7 were detected (Tables 1 and S4). These data are consistent with a role of CDPK4 in activation of the pre-replicative complex (Invergo et al, 2017), and this role may explain the complete lack of DNA replication in cdpk4 deletion mutants (Tewari et al, 2010). In higher eukaryotes, phosphorylation of ORCs is facilitated by CDKs (Lee et al, 2012), whereas Cdc7-Dbf4 PK (DDK) promotes the assembly of a stable Cdc45-MCM complex (Sheu & Stillman, 2006). No homologues of either Cdc7, Dbf4, or Cdc45 have been identified in the Plasmodium genome, and therefore, we suggest that MAP2 performs the function of DDK. MCM2, MCM4, and MCM7 were also co-precipitated with SRPK1-GFP (Tables 1 and S3). Live-cell imaging of male gametocytes suggested that SRPK1-GFP is largely excluded from the nucleus (Fig 1B), but by immunoprecipitation, we detected interactions between SRPK1-GFP and proteins known to be generally found in the nucleus, such as MCM proteins. MCM proteins shuttle between the nucleus and cytoplasm, and it has been suggested that they interact with protein kinases and protein phosphatases in the cytoplasm (Abe et al, 2012). Thus, an interaction between SRPK1-GFP and MCM proteins may occur in the cytoplasm. Phosphorylation of MCM2 is critical for its loading onto chromatin (Chuang et al, 2009), so it is possible that CDPK4 is critical for the association of the ORC with chromatin and the replication origin, whereas SRPK1 and MAP2 facilitate activation of the MCM complex. CDPK4 is also thought to function not only as a kinase but also as a scaffold protein, forming complexes with other proteins such as ORC subunits or CDC6 (Fang et al, 2017), and promoting the interaction between a phosphorylation product and its binding protein (Popova et al, 2018). An altered phosphopeptide profile was only observed in the srpk1 deletion mutant for MCM5 and in the cdpk4 mutant for ORC1 (Invergo et al, 2017), so our hypothesis differs somewhat from that of Fang et al (2017), who identified CDPK4 as part of the MCM complex in non-activated gametocytes. However, in the current study gametocytes were analysed 6 min post-activation, a time point at which MCM function may be regulated by SRPK1 or MAP2, rather than CDPK4.

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Figure 3. Interactome of CDPK4, SRPK1, and MAP2 during male gametogenesis.

(A) Venn diagram showing common interacting partners in gametocytes activated for 6 min, with some additional proteins specific to PK. Each number represents the number of identified proteins. (B, C, D) GO enrichment analysis of CDPK4-GFP (B), SRPK1-GFP (C), and MAP2-GFP (D). BP, biological process; CC, cellular component; MF, molecular function. −log10 P-values obtained from the Bonferroni-adjusted P-values. See also Tables 1, S2, S3, S4, S5, S6, and S7.

MAP2-GFP complexes were also enriched with several MTOC-associated proteins involved in axoneme development including Kinesin-8B, Kinesin-13, and Kinesin-15 and PF16 (Fig 3D, Tables 1 and S4; Straschil et al, 2010; Zeeshan et al, 2019, 2022b). Our results are in partial agreement with those of Fang et al (2017); Kumar et al (2021), who found altered phosphopeptide abundance for all three kinesins in both Pbcdpk4 and Pbsrpk1 deletion mutants (Fang et al, 2017), and Pfcdpk4 mutants (Kumar et al, 2021), respectively. In addition, SMC2/4 was co-precipitated with both CDPK4 and MAP2, whereas SMC1/3 was co-precipitated with SRPK1 and MAP2. SMC1 and SMC3 are part of the cohesin complex that facilitates sister-chromatid cohesion during mitosis, whereas SMC2/4 are part of the condensin complex that regulates chromosome condensation and segregation during cell division (Uhlmann, 2016). Knockdown of smc2/4 significantly affects male exflagellation and parasite transmission (Pandey et al, 2020). In yeast, SMC4 activity and dynamic binding of the condensation machinery are governed by Cdk1 (Robellet et al, 2015); thus, it is possible that a major regulator of condensin activity may be CDPK4 because it interacts with SMC4 (Fang et al, 2017). Our study also suggests that SMC2/4 play a role late in male gametogenesis through interaction with MAP2-GFP, possibly a role in atypical chromosome condensation through association with the MTOC (Guttery et al, 2012).

SRPK1-GFP precipitates were strongly enriched for the translation pathway and 80S ribosome proteins, including 18 40S and 20 60S ribosomal proteins, and several subunits of the eukaryotic translation initiation factor 3 (eIF3) complex (Fig 3C, Tables 1 and S6). mRNA translation, translation elongation, and termination are highly coordinated processes facilitated by the 80S ribosome and an extensive array of eIFs, including eIF3 (Kramer et al, 2019). Co-precipitation of these elements was not unexpected because of SRPK1’s known role in mRNA splicing and translation (Dixit et al, 2010) and suggests that this process, including phosphorylation of the eIF3 complex (Farley et al, 2011), is highly conserved in Apicomplexa. SRPK1 also co-precipitated the co-chaperone HSP40, a protein that mediates dynamic interactions of SRPK1 with the major molecular chaperones HSP70 and HSP90 in mammalian cells (Zhong et al, 2009), and proteins that were detected here (Table 1). However, unlike the findings of other studies (Dixit et al, 2010), we observed no interaction of SRPK1 with serine/arginine-rich (SR) proteins.

Finally, our data are consistent with a role of reversible protein phosphorylation as a key driver of male gametogenesis. CDPK4 and SRPK1 co-precipitated each other (Table 1), consistent with a potential feedback mechanism between these PKs, as suggested previously (Invergo et al, 2017). We also observed co-precipitation of CDPK4 and MAP2 with PPM11, a novel PP shown previously to play a role in male gametogenesis (Balestra et al, 2020). As we also found, altered phosphopeptide abundance was observed for PPM11 in cdpk4 but not srpk1 mutants (Fang et al, 2017), which suggests that PPM11 has a role in mitotic entry and exit through dephosphorylation of CDPK4 and MAP2, respectively. CDPK4 function may also be regulated by calcineurin and PP5 (Table 1), both of which have been implicated in male gametogenesis (Philip & Waters, 2015; Zhu et al, 2019).

Ultrastructure analysis reveals defects in MTOC formation, kinetochore dynamics, and axoneme formation in PK mutants

To determine the ultrastructural defects resulting from loss of these PKs, we used male gametocytes of cdpk4, srpk1, and map2 deletion mutant lines (Tewari et al, 2010) activated for 8 min and examined using ultrastructure expansion microscopy (U-ExM; Gambarotto et al, 2019), and activated for 8 or 15 min and examined using transmission electron microscopy (Fig 4).

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Figure 4. Ultrastructure analysis of WT and cdpk4, srpk1, and map2 gene–knockout male gametocytes.

(A, B) Expansion microscopy images of WT and cdpk4-, srpk1-, and map2-knockout male gametocytes at 8 min post-activation. (A) Maximum intensity projections of whole-cell z-stack images labelled with NHS-ester (grey), α-tubulin (green), and Hoechst (blue). A magenta arrowhead indicates one of the MTOCs (WT, CDPK4-KO, and MAP2-KO) or clustered basal bodies (SRPK1-KO). Scale bar = 5 μm. (A, B) Single z-stack image focusing on the MTOC (WT, CDPK4-KO, and MAP2-KO) or clustered basal body (SRPK1-KO) region highlighted with a magenta arrowhead in (A), displaying NHS-ester (grey), α-tubulin (green), and Hoechst (DNA, blue). N, nucleus; BB, basal body; IM, inner spindle MTOC; OM, outer basal body MTOC. Scale bar = 2 μm. (C) Transmission electron microscopy images of WT and cdpk4-, srpk1-, and map2-knockout male gametocytes at 8 min (a, b, c, e, f, g, i, j, k, m, n, o) or 15 min (d, h, i, p) post-activation. (b, f, j, n) are magnified views of the areas enclosed by the magenta squares in (a, e, i, m), respectively. N, nucleus; K, kinetochore; A, axoneme; BB, basal body; IM, inner spindle MTOC; Mt, microtubule.

In the cdpk4 mutant, U-ExM revealed a single bipartite MTOC in the nucleus, together with a short microtubular structure extending from the MTOC (Figs 4A and B and S4A and B). A similar single bipartite MTOC was observed by U-ExM of CDPK4-KO male gametocytes in a previous study, and centrin was shown to localise to the outer basal body MTOC (Rashpa & Brochet, 2022). Normal development and location of the bipartite MTOC, basal bodies, kinetochores, axonemes, and microtubules revealed in WT parasites by electron microscopy (Fig 4Ca–d) were disrupted in the cdpk4 mutant with some kinetochores appearing to remain within the nucleoplasm (Fig 4Ce), and several attached to the spindle radiating from the MTOC (Fig 4Cf). Dispersed doublet microtubules were present in the cytoplasm, which are likely precursors of axonemes that had failed to form (Figs 4Cf–h and S5C, D, G, and H). These results are like those of Rashpa & Brochet (2022), who had suggested that cdpk4 mutants form partial microtubules from the MTOC. Although CDPK4 interacts with multiple proteins, this phenotype is reminiscent of that of a Kinesin-13–deficient line (Zeeshan et al, 2022b), consistent with our finding that CDPK4 interacts with Kinesin-13 (Table 1). However, our immunoprecipitation data show that CDPK4 interacts with multiple proteins, and its phosphorylation cascade is considered to be complex (Invergo et al, 2017). Therefore, not only CDPK4 and Kinesin-13, but also multiple other proteins may interact to enable spindle assembly, DNA segregation, and axoneme formation during male gametogenesis as suggested in previous studies (Fang et al, 2017; Invergo et al, 2017; Kumar et al, 2022).

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Figure S4. Additional expansion microscopy images of cdpk4, srpk1, and map2 gene–knockout male gametocytes.

(A, B) Expansion microscopy images of cdpk4-, srpk1-, and map2-knockout male gametocytes at 8 min post-activation. (A) Maximum intensity projections of z-stack images labelled with NHS-ester (grey), α-tubulin (green), and Hoechst (DNA, blue). A magenta arrowhead indicates one of the MTOCs (CDPK4-KO and MAP2-KO) or clustered basal bodies (SRPK1-KO). Scale bar = 5 μm. (A, B) Single z-stack image focusing on the MTOC (CDPK4- and MAP2-KO) or clustered basal body (SRPK1-KO) region highlighted with a magenta arrowhead in (A), displaying NHS-ester (grey), α-tubulin (green), and Hoechst (DNA, blue). N, nucleus; BB, basal body; IM, inner spindle MTOC; OM, outer basal body MTOC. Scale bar = 2 μm.

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Figure S5. Additional transmission electron microscopy images of cdpk4-, srpk1-, and map2-knockout male gametocytes.

Transmission electron microscopy images of cdpk4-, srpk1-, and map2-knockout post-activation male gametocytes. (A, E, I, M, Q, U) are whole views of cells. (B, F, J, N, R, V) are magnified views of the areas enclosed by the magenta squares in (A, E, I, M, Q, U), respectively. (C, D, G, H, K, L, O, P, S, T, W, X) are respectively cross-sectional and longitudinal views of either axonemes or microtubules. N, nucleus; K, kinetochore; A, axoneme; BB, basal body; IM, inner spindle MTOC; Mt, microtubule.

The srpk1 mutant lacked MTOCs associated with the nucleus (Figs 4A and S4A and B), and kinetochores were located near the centre of the nucleoplasm (Figs 4Ci and j and S5I, J, M, and N). Whereas in WT cells, all kinetochores (29 of 29 kinetochores from 12 cells) were associated with spindles, in SRPK1-KO cells, no kinetochores (0 of 20 kinetochores from 10 cells) were associated with spindles and all kinetochores were separated from the nucleus periphery (Figs 4Ci and j and S5I, J, M, and N). In SRPK1-KO cells, abnormal basal bodies formed clusters away from the nucleus (Figs 4A and B and S4A), together with partially formed, abnormal axonemes lacking the usual 9+2 WT tubulin subunit configuration (Figs 4Cc, d, k, and l and S5K, L, O, and P). Similar observations were made by Rashpa & Brochet (2022), who found that CDPK4 and SRPK1 are key for axoneme assembly, microtubule nucleation, and development of the MTOC. The loss of the connection between the spindle and the basal body/outer MTOC is a phenotype also observed in Toxoplasma tachyzoites after the knockout or knockdown of the kinetochore and its associated proteins (Farrell & Gubbels, 2014; Brusini et al, 2022; Li et al, 2024). However, the unique MTOC formation and function in Plasmodium gametocytes are likely achieved through complex interactions between various proteins, including kinetochore components and PKs such as CDPK4 and SRPK1.

We showed previously that map2 deletion results in defective chromosome condensation with arrested kinetochore development during male gametogenesis (Guttery et al, 2012). In a map2 mutant line, the two bipartite MTOCs remained adjacent without separation (62% [21/34] of MTOCs from six cells; Figs 4A–Cn and S4A and B), whereas in WT cells, they were dispersed throughout the cytoplasm (100% [28/28] of MTOCs from five cells; Fig 4A–Ca and b). Axoneme structure in a MAP2-KO line appeared to be similar to that in WT cells (Fig 4A and B), with the classical 9+2 subunit arrangement (Figs 4Cm–p and S5S, T, W, and X). However, despite the formation of apparent intact axonemes, exflagellation was not observed. We suggested previously (Guttery et al, 2012) that MAP2 is involved in axoneme function, and impaired axoneme movement may prevent exflagellation. The MTOC separation that is also affected in the map2 mutant may be another reason why gametes are unable to undergo exflagellation. The phenotype resembles that of other mutants during male gametogenesis like crk5, kinesin-8B, kinesin-13, and cdc20 gene knockouts (Guttery et al, 2012; Zeeshan et al, 2019, 2022b; Balestra et al, 2020). More recently, we also observed similar chromosome segregation defects during male gametogenesis, like those seen in cdpk4- and srpk1-knockout lines, in a nek1 knockdown line (Zeeshan et al, 2024). This suggests that these mitotic kinases drive MTOC organisation and chromosome segregation. It will be interesting to study the three-dimensional organisation of these mutants during male gametogenesis using 3D electron microscopy such as serial block-face scanning electron microscopy (Hair et al, 2023).

In conclusion, this study focused on three divergent protein kinases, CDPK4, SRPK1, and MAP2, defined as male-specific because their deletion affects only male gametogenesis. The principal steps in male gametogenesis and the consequence of cdpk4, srpk1, and map2 deletion are summarised in Fig 5. We identified their location during schizogony and male gametogenesis by fluorescence live-cell imaging, and used immunoprecipitation and mass spectrometry to identify binding partners that are potential substrates. Using expansion and electron microscopy, we detailed the ultrastructural defects during male gametogenesis in cells for which each of the genes has been deleted. Our findings provide new insights into the location and function of these PKs and their interacting partners during male gametogenesis, which may underpin the development of drugs targeting this critical stage of the Plasmodium life cycle.

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Figure 5. Schematic diagram of the principal steps in male gametogenesis and the consequence of cdpk4, srpk1, and map2 deletion.

The top diagram illustrates Plasmodium male gametogenesis with the arrow below showing the main events over time in WT parasites. The activated gametocyte is haploid (N). MTOC nucleation occurs in approximately the first 1–2 min after activation. Between 2 and 4 min after activation, the first round of DNA replication (2N: diploid) takes place, the first MTOC replication and separation occurs, and axoneme synthesis begins. Within 4–6 min, the second round of DNA replication (4N: tetraploid) and MTOC replication and separation occur, and axoneme assembly continues. By 8 min post-activation, the third round of DNA replication (8N: octoploid) and MTOC replication and separation occur and axoneme synthesis is completed. Exflagellation begins, and by 15 min post-activation, 8 flagellated gametes (N) bud off from the cell body. The lower arrows indicate the progression of gametogenesis in three different protein kinase gene–knockout parasites, with the red bars indicating the proposed blocking points. In CDPK4-KO parasites, only MTOC nucleation occurs, whereas in SRPK1-KO parasites, complete axoneme assembly is blocked. In MAP2-KO parasites, axoneme assembly is completed, but exflagellation is blocked.